Protein synthesis is a fundamental biological process that underlies the development of polypeptide therapeutics, diagnostics, and industrial enzymes. With the advent of recombinant DNA (rDNA) technology, it has become possible to harness the catalytic machinery of the cell to produce a desired protein. This can be achieved within the cellular environment or in vitro using extracts derived from cells.
Cell-free protein synthesis offers several advantages over in vivo protein expression methods. Cell-free systems can direct most, if not all, of the metabolic resources of the cell towards the exclusive production of one protein. Moreover, the lack of a cell wall in vitro is advantageous since it allows for control of the synthesis environment. For example, tRNA levels can be changed to reflect the codon usage of genes being expressed. The redox potential, pH, or ionic strength can also be altered with greater flexibility than in vivo since we are not concerned about cell growth or viability. Furthermore, direct recovery of purified, properly folded protein products can be easily achieved.
In vitro translation is also recognized for its ability to incorporate unnatural and isotope-labeled amino acids as well as its capability to produce proteins that are unstable, insoluble, or cytotoxic in vivo. In addition, cell-free protein synthesis may play a role in revolutionizing protein engineering and proteomic screening technologies. The cell-free method bypasses the laborious processes required for cloning and transforming cells for the expression of new gene products in vivo, and is becoming a platform technology for this field.
Among the proteins of interest for cell-free synthesis, many either span or are anchored to membranes. These proteins and other biomolecules incorporated into membranes surrounding cells and organelles moderate a wide variety of cellular functions. Furthermore, many lipids and glycolipids are targets for, or activate, protein functions.
For example, about half of potential pharmaceutical targets are membrane proteins, e.g. ion channels and G protein coupled receptors, which have been difficult to utilize in drug screening and design assays. Because of their location in membranes, these proteins are frequently difficult to purify and characterize. They are also difficult to obtain in large quantities, and recombinant DNA methods often fail to provide large amounts of properly folded membrane proteins, in part because overexpression of membrane proteins is generally toxic to living cells, thus limiting the yield.
To avoid this toxicity, in vitro techniques have been attempted to produce higher yields of protein. However, reports have shown either low yields, similar or less than those obtained in vivo; high yields, but where most of the protein is aggregated and must be refolded post-translationally; or high yields where the protein has been synthesized into detergents or liposomes without using the natural folding pathway, raising a question of whether these systems produce membrane proteins that are authentically folded.
One of the major limitations in studying membrane proteins has been the general difficulty in producing significant quantities of correctly folded protein. The present invention addresses this need.
Relevant Literature
U.S. Pat. No. 6,337,191 B1; Swartz et al. U.S. Patent Published Application 20040209321; Swartz et al. International Published Application WO 2004/016778; Swartz et al. U.S. Patent Published Application 2005-0054032-A1; Swartz et al. U.S. Patent Published Application 2005-0054044-A1; Swartz et al. International Published Application WO 2005/052117. Calhoun and Swartz (2005) Biotechnol Bioeng 90(5):606-13; Jewett and Swartz (2004) Biotechnol Bioeng 86(1):19-26; Jewett et al. (2002) Prokaryotic Systems for In Vitro Expression. In: Weiner M, Lu Q, editors. Gene cloning and expression technologies. Westborough, Mass.: Eaton Publishing. p 391-411; Lin et al. (2005) Biotechnol Bioeng 89(2): 148-56.